2.2. Immunohistochemistry of human and mouse cornea

Normal human corneal tissues were obtained from the Eversight eye banks (Ann Arbor, MI, USA) and embedded in paraffin blocks. Immunohistochemical (IHC) staining of human and Ace2^−/− mouse eyes were conducted as described previously. 34 Antigen retrieval of the paraffin embedded sections was performed at 96°C in pH 6.0 citrate buffer for 30min. After blocking in 2.5% normal horse serum, sections were incubated overnight at 4°C with antibodies recognizing: AngiotensinII (AngII) rabbit polyclonal antibody (Penlabs, San Carlos, CA), ACE2 goat polyclonal antibody(R&D systems, Minneapolis, MN), CD68 rabbit polyclonal antibody (Proteintech, Chicago, IL), CD3 rabbit polyclonal antibody (Proteintech, Chicago, IL), and CD11c rabbit polyclonal antibody (Proteintech, Chicago, IL) at 1:100 dilution. After rinsing with PBS containing 0.1% Tween (PBST), the samples were incubated with ImmPRESS‐AP (alkaline phosphatase) Polymer Anti‐Rabbit or Anti‐Goat IgG Reagent (Vector lab, Burlingame, CA) at room temperature for 30 minutes. After rinsing with PBST, chromagen was detected using a Vector Red Alkaline Phosphatase Substrate Kit (Vector lab, Burlingame, CA) for 10‐30 minutes. Samples were counterstained with hematoxylin, and dehydrated with graded ethanol and xylene. Images were taken using an AxioVision Z1 fluorescence microscope system (Carl Zeiss, Oberkochen, Germany).